CN104651319B - Fusarium graminearum low-toxicity virus FgHV2/JS16 and application thereof - Google Patents

Abstract

The invention discloses fusarium graminearum low-toxicity virus FgHV2/JS16. The whole genome sequence of the fusarium graminearum low-toxicity virus FgHV2/JS16 is shown in SEQ ID NO:1 in a sequence table. The fusarium graminearum low-toxicity virus FgHV2/JS16 disclosed by the invention has a remarkable effect for weakening the pathogenicity of fusarium graminearum.

Description

A kind of Fusarium graminearum hypovirus FgHV2/JS16 and its application

Technical field

The present invention relates to biology field, more particularly to a kind of Fusarium graminearum hypovirus FgHV2/JS16 and Its application.

Background technology

Fusarium graminearum (Fusarium graminearum Schw.) is an important kind in sickle-like bacteria, can cause field Between gramineous crop disease.If any wheat scab.After infecting wheat or corn, white flock or velvet-like mycelia are born, be in afterwards White is to rosiness, white to pink or white to brick-red.Perfect stage belongs to for Gibberella zeae Schw.petch Deuteromycotina fungi.Invisible element belongs to Ascomycotina fungi.White aerial hyphae is dense in the medium, substrate mycelium secretion Bronzing pigment.The history of life includes sexual generation and asexual generation.Sexual generation produces the shell of ascus, and pyriform has mouth.Bar-shaped ascus Grow thickly and the shell of ascus, there are eight ascospores in each ascus, ascospore spindle generally there are three barrier films.Asexual generation Conidium is produced, in sickle shaped, generally there are three to five barrier films.

The host range of Fusarium graminearum extensively, can infect cereal crops wheat, barley and corn (Bluhm et Al.2007), and some toxic substances --- trichothecenes (Trichothecenes) and mould can be secreted Toxin estrogen (Oestrogenic mycotoxin), corn germ toxin (Zearelanone), contain certain level toxin Cereal can cause obvious poisoning symptom after by people and animals, including Nausea and vomiting is even miscarried etc..In recent years, differently The sociales bacterium for causing gibberella zeaze petch of wheat and barley in area and different cultivars is Fusarium graminearum (Fusarium graminearum schw) At present, the preventing and treating of the gibberella zeaze petch of wheat and barley that Fusarium graminearum causes is main based on chemical prevention, more using broad-spectrum germicide such as Amici reaches, probenazole and carbendazim etc..Chemical agent is poor to the prevention effect of head blight, it is difficult to retrieve a loss.In morbidity Serious field, can cause the substantial amounts of underproduction.And it is heavy dose of frequently using chemical pesticide can to natural environment including soil, Lower water etc. causes serious pollution.Medicament residue can gradually be assembled into the human body with food chain.The pollution of soil and groundwater The continuous deterioration of natural environment can be caused, the life condition of the mankind is influenceed.Second prevention and controls is mainly farming management.As wanted Rotation cropping, prevents from causing because of continuous cropping the accumulation of pathogen.Carry out soil, cultivation matrix sterilization；During cultivation find diseased plant should and When pull out and destroy, reduce germ further diffusion.Plant culture density is also grasped, strengthens ventilated, reduced wet Degree, controls the water content of soil or matrix, preferably from well-drained matrix.These measures a certain degree of can prevent and treat red The great outburst of mildew and prevalence.

The relatively effective method of controlling disease is cultivation disease-resistant variety.But so far, resisting completely also without commercialization Sick or completely immune wheat breed.Wheat is divided into five classes to the resistance of head blight, and a respectively type is anti-invasion, the anti-expansion of two types Exhibition, three type antitoxins are accumulated, four type seeds are anti-infects and five types of the resistance to sick type of five types " (Mesterhazy et al., 1995).At present, single-gene transformed wheat cultivates transgenic wheat kind and technically has been able to realize (Dahleen et al.2001).Some identified anti gibberellic disease genes are transferred in excellent wheat breed can effectively be improved to head blight Resistance.Host's anti gibberellic disease identified for genes limited amount such as wheat limits the cultivation of disease-resistant variety.The resistance base identified Because what is assembled including the digestive enzyme such as dextranase and chitinase and some thaumatin Ts and obstruction deoxynivalenol Albumen (Anand et al.2003).

Mycoviruses are present in all of main groups fungi.After being reported from first case mycoviruses in 1962, Substantial amounts of mycoviruses are found and study.But the research of mycoviruses does not obtain enough attention also, and shows Certain hysteresis quality.Main cause includes, first, mycoviruses are infected and do not show obvious symptom, therefore are not easy by people Excavate and pay attention to, and most mycologist not will recognize that to be probably due to true when the bacterial strain of bacterium colony exception is touched Caused by bacterium virus infection.Second, the virologist for studying mycoviruses is fewer.Current study hotspot is concentrated mainly on energy Weaken the pathogenic mycoviruses of host, explore these viral replicanisms, protein function and in agricultural fungal diseases life Application in thing preventing and treating.Up to now in mycoviruses system, only chestnut vaccine hypovirus CHV1/EP713 is employed successfully in Biological control, it finds and applies in Italy, has saved the endangered European chestnut woods because of blight of chestnut harm.At this stage The preventing and treating of fungal diseases of plants is a problem, with a large amount of administrations of chemical pesticide, is made to environment while disease control Into huge pollution, including soil pollution, food pollution, atmosphere pollution and water pollution.Mycoviruses are used as a biological control Resource, it will for the preventing and treating of fungal disease provides a new approach.

The content of the invention

The technical problem to be solved in the present invention is to provide one kind and is weakening the pathogenic aspect of Fusarium graminearum with significantly effect The Fusarium graminearum hypovirus FgHV2/JS16 of fruit.

A kind of Fusarium graminearum hypovirus FgHV2/JS16, SEQ ID NO in its whole genome sequence such as sequence table:1 It is shown.

Fusarium graminearum hypovirus FgHV2/JS16 of the present invention is weakening the pathogenic application of Fusarium graminearum.

A kind of bacterial strain, it contains Fusarium graminearum hypovirus FgHV2/JS16 of the present invention.

Bacterial strain of the present invention, preserving number CGMCC NO.10323, preservation date：On January 20th, 2015.

Fusarium graminearum hypovirus FgHV2/JS16 differences from prior art of the present invention are：It is of the present invention Fusarium graminearum JS16 bacterial strains are that this laboratory occurs to be separated on the wheatear of head blight from Jiangsu Province, in 2015 1 The moon has carried out preservation on 20th in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), registers on the books Numbering preserving number CGMCC NO.10323.This laboratory has succeeded from JS16 identification of strains to hypovirus coe virus FgHV2/ JS16, it can weaken the pathogenic functional study of Fusarium graminearum, with the application potential in biological control, and with wide Wealthy application prospect.

Fusarium graminearum hypovirus FgHV2/JS16 of the invention is described further below in conjunction with the accompanying drawings.

Brief description of the drawings

Fig. 1 is by the double-stranded RNA extracted in Fusarium graminearum bacterial strain JS16 in the present invention；Swimming lane M, DNA marker；Swimming Road 1, the dsRNA that bacterial strain JS16 is separated to；Swimming lane 2, bacterial strain HN10 extracts dsRNA as positive control；Swimming lane 2, bacterial strain JS16- F extracts dsRNA as negative control；

Fig. 2 is the genome structure figure of bacterial strain JS16 extraction viruses FgHV2/JS16 in the present invention, and gRNA is viral gene Group structure chart, D-RNA is the structure chart of deficient rna contained by virus；

Fig. 3 is bacterial strain JS16-F Northern hybridization confirmation detoxification situations in the present invention；GRNA is viral genome RNA, D-RNA are the deficient rna of virus, and Prob1 is the end of virus genome RNA 5 ' the cDNA probes of digoxigenin labeled, Prob2 is the end of virus genome RNA 3 ' the cDNA probes of digoxigenin labeled, and JS16 is band toxic bacterial strain, and JS16-F is detoxification bacterium Strain；

Fig. 4 is the colonial morphology of bacterial strain JS16 and JS16-F in PDA culture medium in the present invention；JS16 is band toxic bacterial strain, JS16-F is detoxification bacterial strain；

Fig. 5 be colony diameter (A) in PDA culture medium of bacterial strain JS16 and JS16-F in the present invention, the speed of growth (B) and Biomass (C) on PDB culture mediums；JS16 is band toxic bacterial strain, and JS16-F is detoxification bacterial strain；

Fig. 6 is the conidium Yield comparison of bacterial strain JS16 and JS16-F in the present invention.JS16 is band toxic bacterial strain, JS16-F It is detoxification bacterial strain；

Fig. 7 is that the DON Output of toxin of bacterial strain JS16 and JS16-F in the present invention compares；JS16 is band toxic bacterial strain, JS16-F It is detoxification bacterial strain；

Fig. 8 is the pathogenic comparing of bacterial strain JS16 and JS16-F in the present invention, and A is every wheat head morbidity spikelet number statistics, and B is Wheat head incidence, C is the grain quality situation of morbidity small ear；JS16 is band toxic bacterial strain, and JS16-F is detoxification bacterial strain.

Specific embodiment

The Fusarium graminearum JS16 bacterial strains virus dsRNA of embodiment 1 is extracted

The reaping hook bacteria strain that will first be frozen in glycerol tube is activated.The bacteria cake of picking activated strains, is positioned over covering There are one layer of middle position of the PDA culture medium upper flat plate of glassine paper, 25 DEG C of culture 96h.Viral genome extracting method is with reference to plant The method that thing virus dsRNA is extracted.RNA extract during as far as possible use disposable plastic vessel, if with vessel such as grinding rod, Glassware etc. should as follows carry out treatment and see using preceding, with 0.1%DEPC (pyrocarbonic acid diethyl ester) aqueous solution 37 Processed at DEG C 12 hours, the DEPC that then autoclaving is remained for 30 minutes with removing at 120 DEG C.The key step of experimental implementation It is as follows：1. the glassine paper that mycelia has covered with whole PDA plate is taken off, is put into 2ml centrifuge tubes after gently crimping, use grinding rod Liquid nitrogen grinding is uniform, adds 2 times of 1 × STE buffer solutions of volume (w/v), (v/v) Tris saturations phenol of 2 times of volumes and 2% 10%SDS, is stirred at room temperature 1h；2. supernatant is taken after 10,000rpm centrifugations 20min, adds isometric chloroform, be stirred at room temperature 10min；3. supernatant is taken after 12,000rpm centrifugations 10min, adds ethanol to final concentration of 16% (v/v), add 4% CF- 11 celluloses (w/v).30min is stirred at room temperature；4. 12,000, rpm centrifugation 3min outwells supernatant and stays precipitation, adds and contains 16% ethanol 1 × STE buffer solutions, acutely shake 1min, 1 × STE buffer solution repeated washing three time of the precipitation containing 16% ethanol；⑤12, 000, rpm centrifugation 3min stays precipitation, adds 1 × STE buffer solutions, acutely shakes 1min, 12,000, rpm centrifugation 3min, by supernatant It is transferred in another centrifuge tube, precipitation repeats dissolving once with 1 × STE buffer solutions.Merge the eluent of gained twice；6. add The NaAc (3mol/L, pH 5.2) of 1/10 volume, adds 2.5 times of absolute ethyl alcohols of volume, after mixing, is placed in -80 DEG C of refrigerators 1h；7. 4 DEG C, 12,000rpm centrifugation 15min abandon supernatant and stay precipitation, are washed twice with 70 cold ethanol, are dissolved in 30ul's after drying The ddH2O of DEPC treatment；8. dnase digestion.Add 10 × DNase I of the DNase I and 3.44ul of 1ul RNase-Free After buffer, 37 DEG C of digestion 30min；9. 1% agarose gel electrophoresis.

As a result：With toxic bacterial strain JS16 and its entrained dsRNA viruses through the postdigestive 1% Ago-Gel electricity of DNase As shown in figure 1, virus contains 2 dsRNA, one is more than 10kb, size 5kb or so for swimming.

JS16 is general in China Committee for Culture Collection of Microorganisms on January 20th, 2015 for present invention band toxic bacterial strain Logical microorganism center (CGMCC) has carried out preservation, numbering of registering on the books preserving number CGMCC NO.10323.

The cDNA clone of the Fusarium graminearum hypovirus FgHV2/JS16 genomes of embodiment 2 and sequence analysis

(1) bacterial strain JS16 viral genomes random primer cDNA clone

Random primed reverse transcription:Template is to extract the dsRNA of bacterial strain JS16, and reverse transcriptase is the M-MLV of promega, institute It is Random Primer (26-mer) (5 '-GACGTCCAGATCGCGAATTCNNNNNN-3 ') with primer.

PCR is expanded：The cDNA products for taking previous step synthesis are template, are expanded using single primer, and amplimer is Random Primer(20-mer)(5’-GACGTCCAGATCGCGAATTC-3’).Amplification enzyme used is the 2 of Quan Shi King Companies ×TransScriptTM HiFi PCR SuperMixⅡ。

PCR primer and the connection of carrier T, convert and sequence verification：Using the DNA gel QIAquick Gel Extraction Kit of OMEGA companies, Purifying recovery is carried out to above-mentioned PCR primer to specifications.Purpose fragment is connected to T cloning vectors pMD19-T after recovery (TaKaRa)。

(2) bacterial strain JS16 viral genomes special primer cDNA clone

Special primer is designed:According to random fragment cloned sequence has been obtained, primer is designed.Primer-design software is primer Premier5, method for designing uses default parameters with reference to software document, parameter.The template of design of primers is two and mutually separates Random fragment, upstream and downstream primer is separately designed on two random fragments.First determine whether that random fragment is during design primer No is positive-sense strand, and second aspect judges the directionality of sequence, and the 3rd aspect judges that the mutual upstream and downstream of random fragment is closed System, i.e., at a distance of virus 5 ' hold or the 3 ' distances held.By the primer between preliminary judgement design random fragment, it is impossible to sentenced Disconnected random sequence, is expanded by designing various possible sequences (including positive, reverse complemental) primer.

PCR is expanded and PCR primer and the connection of carrier T, is converted and checking sequencing:Annealing temperature is according to drawing when PCR is expanded The Tm values of thing determine that general annealing temperature subtracts 5 equal to Tm values.Extension of time determines according to fragment length, because two with Bigpian Sequence length in the middle of section is unknown, so typically setting extension of time more long, 3min.

(3) bacterial strain JS16 viral genomes RACE

Virus 5 ' end sequence amplification:The method of 5 ' end RACE is 5 ' classical RACE cloning process, using terminal deoxy Nucleotidyl transferase (TdT) adds polyC tails at the 3 ' ends of reverse transcription product cDNA, then special using adapter-primer and sequence Different primer carries out nested PCR amplification.PCR primer and the connection of carrier T, convert and checking sequencing.

The end sequence of viral genome 3 ' is expanded：3 ' end sequences amplification technology used is connected for oligo RNA ends RACE(RLM-RACE).In the presence of T4RNA ligases, by a disconnected oligonucleotide chain (5 ' end phosphoric acid with special modification Change modification, the modification of 3 ' Amino End Groups) it is connected to 3 ' C-terminals of RNA chains.According to oligonucleotide chain sequences Design special primer, with 3 ' end sequences together are amplified by 3 ' terminal sequence special primers.PCR primer and the connection of carrier T, convert and checking sequencing.

(4) bacterial strain HN10 virus genome complete sequences splicing

Using the sequence assembly function of DNAMAN softwares by random primer cDNA clone sequence, special primer cDNA clone sequence Row, RACE cloned sequences are stitched together, and constitute complete virus genome sequence.

As a result：The full-length genome of virus entrained by bacterial strain JS16 is 12800nt (referring to SEQUENCE LISTING). It can encode an ORF to DANMAN software predictions, and the ORF originates in base at the 458th, terminates at base at the 12290th, altogether 3944 polyproteins of amino acid of coding, the protease domain comprising virus, helicase domain, the RNA that RNA is relied on gathers Synthase domain (RDRP) and a new SMC domain (Fig. 2).According to genome structure and phylogenetic analysis, by virus The classification position of FgHV2/JS16 is defined as a kind of new virus of hypovirus section.The virus is containing 4553 nucleotides Defective RNA (D-RNA) (Fig. 2).

It is prepared by the detoxification of embodiment 3 strain

(1) with reference to the drug-treated process of Noem í Herrero etc., beaten with card punch and take 5mm with strain pure culture biscuits involvng inoculation in containing It is that band strain, 25 DEG C of lucifuge cultures are processed on 100 μm of Ribavirin PDA plates of ol/L to have concentration；Treat that mycelia will cover with entirely Picking mycelia tip about 0.5mm before plate, is inoculated in same 25 DEG C of lucifuges on containing 100 μm of PDA plates of ol/L Ribavirins Squamous subculture；After mycelia is grown, beaten at mycelia edge and take the pure culture biscuits involvng inoculation of diameter 0.5mm in being 100 μm of ol/L containing concentration In the 100mL CMC culture mediums of Ribavirin, 25 DEG C of lucifuges of 200rpm/min produce spore；Microscopy produces spore situation after 4-5d, uses individual layer Sterilizing Mirocloth filtered through gauze spore liquids, concentration filtrate to concentration be 1 × 108/mL, by 3 × 108 spore inoculatings in 100mL contains during concentration is 100 μm of YEPD culture mediums of ol/L Ribavirins, 25 DEG C, 200rpm/min shaking table cultures 12-14h (here the time to control, the time is too short, and spore germination is insufficient, can be mixed with protoplast during filtering, influence it is primary Plastid quality, the time is oversize, and mycelia is too old, and cell membrane is difficult enzymolysis, influences protoplast yield)；With individual layer Mirocloth yarns Cloth filters mycelia, can wash mycelia with sterilizing ddH2O, in order to avoid not sprouting spore influence follow-up test, mycelia is placed in into sterilizing filter Excessive moisture is sucked on paper, is put into 20mL enzymolysis liquids, 28 DEG C, 90r/min jogs 2h or so, period carries out mirror every 30min Inspection, observes mycelia Degree of Enzymatic Hydrolysis；Observation can stop enzyme digestion reaction after producing a large amount of protoplasts, with 2 layers of Mirocloth gauzes Filtering enzymolysis liquid, collects protoplast, by 4 DEG C of centrifugation 1min of protoplast 2600g/min, supernatant discarded, with precooling on ice STC buffer solutions wash protoplast 3 times by above-mentioned centrifugal condition；It is 400/mL by protoplast diluted concentration, takes 50 μ L paintings It is distributed on regeneration culture medium, 25 DEG C of lucifuge overnight incubations, the picking single bacterium colony under stereomicroscope, it is 100 μ to be inoculated in containing concentration On the PDA plate of mol/L Ribavirins, after mycelia is grown, beat and take 0.5mm bacteria cakes, be inoculated in be covered with sterilizing glassine paper contain Concentration is on 100 μm of PDA plates of ol/L Ribavirins, mycelia to be collected by mycelia is covered with after full plate.

(2) RNA of candidate's detoxification bacterial strain is extracted, with FgHV2/JS16 geneome RNAs 5 ' end and the 3 ' ends of digoxigenin labeled CDNA probes do Northern hybridization with the RNA of candidate's detoxification bacterial strain respectively, verify the authenticity of detoxification.

As a result：Confirmed by the RT-PCR and dot hybridization of bacterial strain, obtain one plant of detoxification bacterial strain, be named as JS16-F (figures 3)。

The biological function of the Fusarium graminearum hypovirus FgHV2/JS16 of embodiment 4

(1) determined with toxic bacterial strain and without toxic bacterial strain colonial morphology and the speed of growth

Beaten with 5mm card punch and take with toxic bacterial strain JS16 and be placed in PDA culture medium flat board without the bacteria cake of toxic bacterial strain JS16-F Center, activated strains；The colony edge bacteria cake for taking activated strains is beaten with 5mm card punch, bacteria cake is transferred to PDA and CM cultures The center of base upper flat plate, respectively three repetitions；Colonial morphology and measurement colony diameter are observed after 25 DEG C of dark culturing 36h and is counted Analysis.Each treatment is in triplicate.

(2) with toxic bacterial strain and without the observation of toxic bacterial strain conidium form and determination of yield

Beaten with 5mm card punch and take with toxic bacterial strain JS16 and be placed in PDA culture medium flat board without the bacteria cake of toxic bacterial strain JS16-F Center, activated strains；The colony edge bacteria cake for taking activated strains is beaten with 5mm card punch, a ferfas cake is transferred to and is equipped with In the triangular flask of 50mlCMC culture mediums, 25 DEG C, 200rpm/min shaking table cultures 5d；Spore suspension is filtered with three layers of lens wiping paper After take filtered fluid examine under a microscope spore shape and with blood counting chamber measure spore concentration, each treatment three repetitions.

(3) with toxic bacterial strain and without toxic bacterial strain Pathogenic Tests

The small weir 22 of plantation wheat, is inoculated with when wheat growth to flowering peak period for wheatear.Now seed does not have also Grow, beneficial to infecting for Fusarium graminearum；Prepare as stated above with toxic bacterial strain JS16 and the conidium without toxic bacterial strain U3, And with blood counting chamber measure spore concentration after spore concentration is adjusted to 3 × 10⁵It is stand-by；Choose the basically identical wheat of growing way Fringe, when finding the 5th small ear from bottom to top, with finger by the inner glume of small ear and coetonium separate, is careful not to injure small ear.With Liquid-transfering gun draw 10ul prepare spore liquid injection inner glume and it is coetonium between root, gently closure keep its nature. Marking pen mark inoculation small ear simultaneously hangs up label on wheat straw, the information such as record inoculating strain, inoculation date, inoculation people.Often Individual bacterial strain is at least inoculated with 15 wheatears；With watering can to water is sprayed into inside freshness protection package, be set on the wheatear of inoculation, it is beneath gently Tie, 25 DEG C of light darks replace (16h/18h) moisturizing culture 48h；Remove freshness protection package and continue culture 14d after, observe wheat Fringe incidence, cuts wheatear and takes pictures and count every fringe morbidity spikelet number.

(4) morbidity seed DON content of toxins is determined

Freeze drying example overnight, weighs 0.5g morbidity seed samples, is transferred to 5ml after being fully ground with liquid nitrogen completely Centrifuge tube is fully evaporated completely all of liquid nitrogen；Add 4ml acetonitrile/water (84/16), on shaking table shake extraction 24h (rotating speed and Temperature is suitable just, without too big requirement)；Take 3ml extracts and cross post (3ml plastics pillars, filling 500mg C18/ neutral oxygens Change aluminium=1/3), collection filtrate is in 1.5ml centrifuge tubes；1ml is in 1.5ml centrifuge tubes for transfer, and 50 DEG C are dried overnight；Freeze-drying Fully (a small amount of water can all have a strong impact on follow-up silanization effect)；Add TMS (TMSI/TMCS=100/1) silicon of 100 μ l Alkylators, abundant vortex centrifugal pipe makes all samples of TMS and tube wall all contact fully；Centrifuge tube is put on oscillator and is shaken 10min is swung, adds the chromatographic grade isooctane of 800 μ l, vortex centrifugal pipe to make fully mixing, add the μ l of ultra-pure water 800, be acutely vortexed Become sample to clarify and be layered；10min (or centrifugation 12000,2min) is stood, supernatant is transferred completely into GC loadings bottle Row concentration mensuration.

As a result：From bacterium colony proterties, mycelial growth, conidium yield, pathogenic and six aspect ratios of DON Output of toxin Compared with the difference with toxic bacterial strain JS16 Yu detoxification bacterial strain U3, experiment proves that virus FgHV2/JS16 influences the bacterium colony proterties of host strain (see Fig. 3)；Substantially suppress mycelial growth, mycelial growth rate reduction by 16%, colony diameter reduces by 33.1%, biomass reduction 47.1% (see Fig. 4)；To conidial yield reduction 70.7% (see Fig. 5)；To the reduction by 77.4% of DON Output of toxin (see figure 6)；Substantially weaken the pathogenic of host Fusarium graminearum, the average onset small ear per wheatear reduces by 44.6% (see Fig. 7).

Embodiment described above is only that the preferred embodiment of the present invention is described, not to model of the invention Enclose and be defined, on the premise of design spirit of the present invention is not departed from, those of ordinary skill in the art are to technical side of the invention Various modifications and improvement that case is made, all should fall into the protection domain of claims of the present invention determination.

Claims (4)

1. a kind of Fusarium graminearum hypovirus FgHV2/JS16, it is characterised in that：SEQ in its whole genome sequence such as sequence table ID NO:Shown in 1.

2. the Fusarium graminearum hypovirus FgHV2/JS16 described in claim 1 weaken Fusarium graminearum it is pathogenic should With.

3. a kind of bacterial strain, it contains the Fusarium graminearum hypovirus FgHV2/JS16 described in claim 1.

4. bacterial strain according to claim 3, preserving number CGMCC NO.10323, preservation date：On January 20th, 2015.